The project continues and significantly extends the development of an innovative strategy for hybridization of oligonucleotides and other probes to double-stranded (ds)DNA. A primary tool is a special class of pyrimidine peptide nucleic acid (PNA; a DNA synthetic mimic) - cationic bis-PNAs or PNA 'openers', known to effectively invade short purine tracts of dsDNA. A major idea underlying the project is that a pair of PNA openers bound to closely located purine sites on dsDNA opens the double helix in between. This makes the DNA target locally accessible for binding the probe via Watson-Crick pairing. Such a complex, the PD-loop and related structures, forms highly sequence-specifically because only the dsDNA site opened in concert by two openers is available for subsequent binding the probe. The goal of the project is to elaborate robust PD-loop based assays applicable to genomic DNA. To reach the project objectives, the sequence limitations intrinsic in the original PD-loop design (only closely located purine sites could be targeted by PNA openers) will be eliminated. Extended PD-loops with shorter openers and longer gaps between purine sites will be designed. PNA openers with intercalators and extra positive charges will be used for their stabilization. Tris-PNA constructs will also be tried as PNA openers advantageous for extended PD-loops. Enhanced-affinity DNA probes and more stable PNA probes will be involved for this purpose, too. PNA openers with the extended triplex recognition will be used for mostly purine sites with few pyrimidines. A pseudocomplementary PNA modification, pcPNA, will be employed as an opener to substantially relieve the PD-loop sequence limitations. A combination of pcPNA openers with pseudocomplementary oligonucleotides invading the mixed purine-pyrimidine dsDNA sequence at the edge of the duplex will finally result in the design of essentially sequence-universal PD-loops. Besides softening the sequence limitations, high sensitivity of DNA diagnostics is required given a small amount of the DNA target relatively to a huge excess of unrelated genomic DNA. Duplex DNA capture with circularized PD-probes (earrings) enabling more stable attachment to target site will be used for enrichment of DNA analytes with the designated PD-target. The rolling-circle hyperamplification (HRCA) of circular probes is expected to provide requisite sensitivity yielding the dsDNA product in which the original PD-site repeats more than million times. Finally, thus multiply copied PD-sites will be selectively exposed by PNA openers and fluorescently detected with molecular beacons. The strategies with simultaneous targeting the PNA openers and molecular beacons to the HRCA-multiplied dsDNA targets for the real-time monitoring of sequence-unrestricted PD-loop hybridization will be elaborated. PD-loop based artificial nickase systems will be designed as an alternative method for site-directed multiple labeling of megabase DNAs. All these strategies will be tested in multiplex detection of dsDNA markers in crude extracts. The implementation of the project will open totally new opportunities for DNA diagnostics of pathogens and for isolation and analysis of genes in an intact form.